USPTO Patents Application 10589866

WO 2005/080414

PCT/IL2005/000199

PEPTIDE MIMOTOPES OF MYCOBACTERIAL MANNOSYLATED

LIPOGLYCANS AND USES THEREOF

FIELD OF THE INVENTION

This invention relates to novel amino acid molecules that mimic epitopes
(mimotopes) of mycbacterial mannosylated lipoglycans such as lipoarabinomannan
(ManLAM) and induce an immune response to ManLAM and their use in
5 diagnosis and immunization against mycobacterial infections.

LIST OF PRIOR ART

The following is a list of prior art considered to be relevant to the subject
matter of the present invention:

Chatterjee, D., et al. J Biol Chem 267: 6234-6239 (1992);
10 Prinzis, S., et al. J Gen Microbiol 139: 2649-2658 (1993);

Nigou, J. Biochimie 85:153-166 (2003);

Hetland G, et al. Clin Diagn Lab Immunol. 5(2):21 1-8 (1998);

Antunes A, et al. Res Microbiol. 153(5):301-5 (2002);

Glatman-Freedman A., FEMS Immunol Med Microbiol. 39(1):9-16
15 (2003).

Hamasur B, et al., Vaccine 16:17(22):2853-61 (1999);

Luo P., et al. J Biol Chem.; 275(21):16146-54 (2000);

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BACKGROUND OF THE INVENTION

Tuberculosis (TB) is mostly a pulmonary lung disease caused by
Mycobacterium tuberculosis (Mtb). This organism is a slow-growing bacillus
that is transmitted by the respiratory route. Soon after infection., the bacilli
5 penetrate alveolar macrophages and survive within early phagosomes. Innate
immune responses directed by macrophages predominate early in the infection.
Subsequent recruitment of dendritic cells leads to cell-mediated responses
involving CD4 + and CD8 + T cells and eventually to granuloma formation.
Among infectious diseases, tuberculosis remains the second leading killer of

10 adults in the world, with more than 2 million TB-related deaths each year (CDC
2004). The vast majority of immunocompetent individuals are able to contain,
but not eliminate, the pathogen in pulmonary granulomas, leading to latent
tuberculosis infection a small minority of cases, through unclear mechanisms,
persistent bacilli can reactivate to produce disease many years to decades after

15 initial infection.

Virulent Mycobacterium cell wall-associated lipoglvcans

A major cell surface component of Mtb and other virulent Mycobacterium
sp. is the polysaccharide mannosylated lipoarabinomannan (ManLAM).
ManLAM is a phosphatidylinositol-anchored lipoglycan composed of a mannan

20 core with oligoarabinosyl-containing side-chains with diverse biological
activities. This polysaccharide accounts for up to 5 mg g -1 bacterial weight.
ManLAM structure differs according to mycobacterial species, and three general
classes of LAM have been described: (i) ManLAM, from the virulent strains
Erdman and H37Rv and the avirulent strains H37Ra and BCG [Chatterjee, D., et

25 al. J Biol Chem 267: 6234-6239 (1992); Prinzis, S., et al. J Gen Microbiol 139:
2649-2658 (1993)], which is characterized by extensive mannose capping of the
arabinan termini; (ii) phospho-myo-inositol-capped LAM (PILAM), found in the
rapidly growing mycobacteria M. smegmatis and M. fortuitum [Nigou, J.
Biochimie 85:153-166 (2003)]; and (iii) AraLAM, which was described in the

30 rapidly growing M. chelonae and lacks mannosylation in its arabinan termini

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[Guerardel, Y. 5 et al. J Biol Chem 277:30635-30648 (2002)]. Although there is
significant heterogeneity between LAM molecules with respect to glycosylation
and acylation [Nigou et al. (2003) ibid.], differences in biological activity
between the major classes of LAM have been attributed primarily to the heavy
5 mannose capping of ManLAM [Chatterjee et al. (1992). ibid.}

In several publications it has been shown that sera of TB patients contain
higher levels of anti-ManLAM antibodies than that of healthy individuals
[Hetland G, et al. Clin Diagn Lab Immunol. 5(2):211-8 (1998)]. Consequently it
has been suggested to be one of the candidate antigens for rapid diagnosis of TB
10 [Antunes A, et al. Res Microbiol. 153(5):301-5 (2002)].

LAMs that are surface components have been suggested as target
molecules for an efficient vaccine candidate due to its presence on the bacterial
surface [Glatman-Freedman A, FEMS Immunol Med Microbiol. 39(1):9-16
(2003)] Nonetheless, purified ManLAM was found to be a poor immunogen
15 [Hamasur B, et al., Vaccine 16:17(22):2853-61 (1999)]. It has been suggested to
conjugate mycobacterial polysaccharides to a carrier protein [Glatman-Freedman
A, (2003) ibid.].

SUMMARY OF THE INVENTION

The present invention is based on the identification, by screening through
20 random phage display libraries, of several peptide-based mannosylated
lipoarabinomannan (ManLAM) mimotopes. This group of peptides showed
selective and direct binding to CS40 anti-ManLAM monoclonal antibody, while
no binding to antibodies against other, non-mannosylated, mycobacterial
lipoglycans, and competitive binding, vs. ManLAM, to CS40 anti-ManLAM
25 antibody. They were also bound by IgG antibodies in sera of TB patients and Mtb
experimentally infected mice, thus higher titers were measured in diseased
patients and animals when compared to healthy immunized and non-immunized
individuals and to naive mice. In addition, the tested peptides were capable of
eliciting production of ManLAM-binding antibodies in vivo.

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Thus, it has been envisaged that these novel peptide-based mimotopes and
the like, may serve as potential amino acid based probes for diagnosis of
mycobactarial infections in subjects characterized by the presence of ManLAM
binding antibodies in the subjects, as well as for immunization of subjects against
5 such mycobacterial infections. It should be noted that the term "mimotope"
[Meloen R. et al. J. Mol Recognit. 13:352-359 (2000)] is also known in the art
by the terms "mimitope" [Arnold G. et al Intervirology 39:72-78 (1996)];
"mimotypes" [Benhar I. Biotechnol Adv. 19:1-33 (2001)]; "mimetics" [Vyas et
al. Proc. Natl Acad Set USA 100:15023-15028 (2003)] and refers to a

10 compound that mimics the structure of an epitope and provokes an identical
antibody response. The compound may be of the same or of a different type of
molecule as the original epitope.

Thus, according to a first of its aspects, the present invention provides an
amino acid molecule comprising a peptide comprising at least one of the

1 5 following characteristics :

(a) being capable of binding to ManLAM binding antibodies;

(b) being capable of eliciting production of ManLAM binding
antibodies.

The invention also provides a method for diagnosing a mycobacterial
20 infection in a subject by determining the presence of ManLAM binding
antibodies in a sample from said subject, the method comprising:

(i) contacting said sample with an amino acid molecule as defined
above;

(ii) determining binding of said amino acid molecule to ManLAM
25 binding antibodies;

wherein a positive determination indicates mycobacterial infection in the
subj ect.

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Within this diagnostic aspect of the invention, there is also provided a kit
for diagnosing mycobacterial infection in a subject, the kit comprising an amino
acid molecule as defined above.

In light of the immunostimulating effect of the amino acid molecules, the
5 invention also provides a vaccine comprising as active agent the amino acid
molecule as defined herein, together with a physiologically acceptable carrier.

According to this aspect of the invention there is also provided a method
of immunizing a subject to mycobacterial infection, the method comprises
providing said subject with an immunizing amount of an amino acid molecule as
1 0 defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out
in practice, a preferred embodiment will now be described, by way of non-
limiting example only, with reference to the accompanying drawings, in which:

15 Figures 1A-1B are graphs showing competitive binding (% binding) vs.

ManLAM of phage clones presenting different peptides (Fig. 1A) or of synthetic
peptides Bll and Al (Fig. IB) to CS40 anti-ManLAM monoclonal antibody.

Figures 2A-2D are bar graphs showing ManLAM-binding antibodies
produced in B 1 1 immunized mice: IgG in phage immunized mice (Fig. 1A) IgM
20 (Fig. IB) IgG (Fig. 1C) and IgG isotypes (Fig. ID) in synthetic, KLH-peptide
immunized mice.

Figure 3 Is a bar graph showing binding of antibodies from sera of 30
days and 3 month Mtb infected mice to ManLAM or Bll mimotope coated
plates, as compared to naive mice.

25 Figure 4 is a graph showing antibodies in TB human patients sera binding

to ManLAM and the Bll mimotope, compared to non-vaccinated healthy
individuals and BCG-vaccinated healthy individuals. Median represented by
triangle. The OD presented is the average OD 405 value of each triplicate sample.

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Figures 5 A-5C are images showing the binding of B 1 1 FITC-labeled
peptide to the macrophage RAW 264.7 cell line as is illustrated by confocal
micrograph (Fig. 5A) and FACS analysis (Fig. 5B), quantifying the fluorescence
levels corresponding to the binding of the Bll FITC-labeled peptide, and the
5 reduction of binding when 0.125 MD-mannose was added (Fig. 5C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery, by phage display
technology, of antigenic and immunogenic peptide-based mimicry of mannose-
containing cell-wall compounds of Mycobacterium tuberculosis, specifically,
10 mannosylated cell wall lipoglycan, such as ManLAM.

Thus, according to a first of its aspects, the invention provides an amino
acid molecule comprising a peptide comprising at least one of the following
characteristics:

(a) being capable of binding (selective binding, direct binding and/or
1 5 competitive binding) to ManLAM binding antibodies;

(b) being capable of eliciting production of ManLAM binding
antibodies.

The term "binding" as defined above refers to at least one of "selective
binding", "direct binding" and "competitive binding" (see bellow). In accordance
20 to the preferred embodiment of the invention this term refers to selective, direct
and competitive binding.

"Selective binding" denotes binding of the amino acid molecule or the
phage presenting the amino acid molecule, to ManLAM binding antibodies while
not binding, at least at a detectable level as determined by methods hitherto
25 known in the art, to antibodies directed to low- and non-mannosylated
polysaccharides.

"Direct binding" denotes the binding of the amino acid molecule directly
to the antigen binding portion of an antibody as determined for example by phage
displaying the amino acid molecule immobilized onto a solid surface such as an

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ELISA plate or a nitrocellulose membrane and then testing for binding thereof to
the antigen binding site of an antibody, either mAb or antibodies in sera of
mycobacterial infected subjects.

"Competitive binding" denotes the dose dependant inhibition of the
5 binding of antibodies (such as CS40 antibodies) to ManLAM by the amino acid
molecule or phage displaying the amino acid molecule.

The term "ManLAM binding antibodies" as used herein denotes
monoclonal antibodies (mAb) as well as polyclonal antibodies which bind to
ManLAM. Preferably, such antibodies bind to lipoglycan carrying relatively high
10 level of mannose residues, such as ManLAM, lipomannan (LM) and
arabinomannan (AM). It is preferable that the antibodies bind at least to
ManLAM.

Binding efficacy of the amino acid molecule to ManLAM binding
antibodies may be evaluated by determining the magnitude of OD decrease when
15 serial dilutions of phage clones/amino acid molecule are incubated with CS40
mAb. The phage clones/amino acid molecule with a higher affinity to mAb will
show maximum inhibition of the binding of the mAb to ManLAM with the
lowest number of phage particles or lowest amount of amino acid molecule,
respectively.

20 The amino acid molecule according to the invention and in particular, the

peptide forming part of the amino acid molecule, may comprise naturally
occurring amino acids, semi-synthetic amino acids as well as synthetic amino
acid sequences.

The term "naturally occurring amino acid" refers to a moiety found
25 within a peptide and is represented by -NH-CHR-CO-, wherein R corresponds to
the side chain of the 20 naturally appearing amino acids.

Nonetheless, the peptide of the invention may also comprise non-naturally
occurring amino acids.

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The term " non-naturally occurring amino acid" (amino acid analog) is
either a peptidomimetic, or is a D or L residue having the following formula:
-NH-CHR-CO-, wherein R is an aliphatic group, a substituted aliphatic group, a
benzyl group, a substituted benzyl group, an aromatic group or a substituted
5 aromatic group and wherein R does not correspond to the side chain of a
naturally-occurring amino acid. This term also refers to the D-amino acid
counteipart of naturally occurring amino acids. Amino acid analogs are well
known in the art; a large number of these analogs are commercially available.
Many times the use of non-naturally occurring amino acids in the peptide has the

10 advantage that the amino acid molecule is more resistant to degradation by
enzymes which fail to recognize them.

A further characteristic of the amino acid molecule of the invention is that
it binds to antibodies which have specificity to mannose containing
polysaccharide. This is evident from their binding to anti-ManLAM antibodies

15 vs. their the lack of binding to antibodies directed against polysaccharides which
do not have mannose residues, such as to CS35 anti-LAM mAb, 735 anti-ploy
a(2^-8) N-acetyl neuraminic acid mAb, and 2H1 anti- glucuronoxylomannan
mAb.

In order to determine whether an amino acid sequence falls within the
20 scope of the invention one of the following assays should be determined:

Assay 1: Binding to ManLAM antibodies:

(a) The selectivity of binding of a candidate sequence by one option may
be examined by binding to the CS40 mAbs against ManLAM while not binding
to other anti-polysaccharide mAbs such as CS35 anti-LAM, 735 anti-poly alpha

25 N-acetyl neuraminic acid mAb and 2H1 anti-glucronoxylomannam mAb-as
described in the detailed description part of the application.

(b) By another option the competitive binding candidate amino acid
molecule may be determined based on the dose dependent inhibition of the
binding of Abs to ManLAM by the candidate amino acid molecule.

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(c) By yet another option, direct binding may be determined based on
direct binding measurements, i.e. determination of ManLAM antibodies, both
monoclonal antibodies and antibodies from individuals' sera exposed to
mycrobactrial infection to the amino acid molecule. The direct binding
5 measurements is based on the fact that antibodies level from mycobacterial
infected subjects is higher that the level thereof, if at all, in healthy subjects
(vaccinated or healthy, non-vaccinated).

The candidate will have one or more of the above binding properties
preferably all three.
10 Assay 2: Eliciting an immune response

According to this assay, a candidate amino acid molecule is preferably
conjugated to a carrier protein and injected to a test animal (in accordance with
the procedure in the detailed description part). The serum of the immunized
animal is tested for the capacity to produce specific ManLAM binding antibodies
15 (e.g. IgG, IgA or IgM) as demonstrated in serological tests.

Candidate amino acid molecules that were active in one of the assays
above fall under the scope of the present invention.

Based on phage display technology six dodecamer peptides were
identified (generally referred to herein as the original amino acid molecules).
20 These peptides are depicted in the following Table 1 :

Table 1: ManLAM peptide mimotopes
Clone Amino acid sequence

WEADDKNQHGEG

(SEQ ID NO:6)

Bll

ISLTEWSMWYRH

(SEQ ID NO :1)

CI

EEGPWSTHVGRT

(SEQ ID NO:2)

C3

WGNEGGDHLQPV

l v ■ %

(SEQ ID NO:3)

F7

SLKIKWELKMYQE

(SEQ ID NO:4)

G3

AVEKWEKHTWSE

(SEQ ID NO:5)

*The postulated motif amino acids are indicated in gray box and bold letters

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Optimal alignment of the above peptides (as described in the Materials
and Methods) identified a motif marked in the above Table 1 in gray.

Interestingly, an internal aromatic amino acid residue was identified in all
these dodeca-peptides. This finding is in correlation with previous evidence
5 suggesting that aromatic residues are critical in peptide sequences that mimic
surface conformations specifically recognized by sugar binding ligands. [Luo P
et al. J Biol Chem.; 275(21): 16146-54. (2000)]. Further, in some of these
peptides, more than one internal aromatic residue was present. For example, B 1 1
comprises two tryptophan and one tyrosine internal residues (...WSMWY...),
10 and F7 and G3 both comprise two aromatic residues (...WELKMY...
and. . . WEKHTW. . ., respectively). Thus, according to one aspect, the amino acid
molecules according to the invention comprise one or more internal aromatic
residues.

Another interesting observation from the above alignment is that an
15 internal aromatic amino acid residue has an adjacent, typically, although not

necessarily, preceding hydrophilic amino acid residue such as glutamate (E),

proline (P), arginine (R).

According to the invention, the amino acid molecule may comprise one or

more copies of the same peptide (monoepitopic peptide constructs) or different
20 peptides (multiple antigenic peptide constructs) and immunological modifications

of the above identified peptides. The amino acid molecules may be linked to

carrier proteins, all as defined herein below.

The "internal aromatic amino acid residue" includes a naturally

occurring and non-naturally occurring amino acid residue carrying an aromatic
25 side chain, the residue being flanked from the amino as well as from the

carboxylic termini thereof by at least on residue (which may naturally occurring,

non-naturally occurring or peptidomimetic organic moiety, all being as defined

herein). For instance, the internal aromatic tryptophan identified in the above

peptides may be replaced with one of the naturally occurring aromatic residues,
30 Phenylalanine (F); Histidine (H), Tryptophan (W), Tyrosine (Y) and conservative

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substitutions thereof, as long as the immunological character of the molecule is
retained.

The term "immunologic modification" according to the invention includes
any one of the following: substitution of one or more amino acid residues,

5 deletion of one or more amino acid residues, insertion of one or more amino acid
residues, chemical modification of one or more amino acid residues, as well as
other modifications known in peptido-based technologies, all of which resulting
in an amino acid molecule which retains the immunologic character of the
original amino acid molecule from which it is derived.

10 The term "immunologic character" concerns both the antigenic character

(i.e. the capacity to bind ManLAM binding antibodies), as well as the
immunogenic character (i.e. the capacity to elicit, upon immunization, production
of ManLAM binding antibodies) of the original amino acid molecule.

Modification also includes alteration of a bond within the peptidic

15 backbone, i.e. that the bond between the N- of one amino acid residue to the C-
of the next has been altered to non-naturally occurring bonds by reduction (to
-CH 2 -NH-), alkylation (methylation) on the nitrogen atom, or the bonds have
been replaced by amidic bond, urea bonds, or sulfonamide bond, etheric bond
(-CH2-O-), thioetheric bond (-CH 2 -S-), or to -CS-NH-; The side chain of the

20 residue may be shifted to the backbone nitrogen to obtain N-alkylated-Gly (a
peptidoid).

Modification also includes cyclization of the amino acid molecule, e.g. by
forming S-S bonds. S-S bonds may be formed via the inclusion of sulphor-
containing amino acid residues, such as cysteine at each terminus of the amino
25 acid molecule. Cyclic peptides have been shown to be more stable and with
higher biological activity than the corresponding linear molecule [Tibbetts S. et
al. Peptides . 2 1 (8) 1 1 6 1 -7 (2000)] .

"Substitution" includes the replacement of one or more amino acid
residues either by other naturally occurring amino acids, (conservative and non-
30 conservative substitutions), by non-naturally occurring amino acids (conservative

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and non-conservative substitutions), or with organic moieties which serve either
as true peptidomimetics (i.e., having the same steric and electrochemical
properties as the replaced amino acid), or merely serve as spacers in lieu of an
amino acid, so as to keep the spatial relations between the amino acid spanning

5 this replaced amino acid.

The term "conservative substitution" in the context of the present invention
refers to the replacement of an original amino acid present in the identified amino
acid molecules with a naturally or non-naturally occurring amino or a
peptidomimetic residue having similar steric properties. Where the side-chain of the

10 original amino acid to be replaced is either polar or hydrophobic, the conservative
substitution should be with a naturally occurring amino acid, a non-naturally
occurring amino acid or with a peptidomimetic moiety which is also polar or
hydrophobic (in addition to having the same steric properties as the side-chain of
the replaced amino acid). However where the original amino acid to be replaced is

15 charged, the conservative substitution according to the definition of the invention
may be with a naturally occurring amino acid, a non-naturally occurring amino acid
or a peptidomimetic moiety which are charged, or with non-charged (polar,
hydrophobic) amino acids that have the same steric properties as the side-chains of
the replaced amino acids. The purpose of such a procedure of maintaining the steric

20 properties but decreasing the charge is to decrease the total charge of the
compound, for example for improving its membrane penetrating properties.

For example in accordance with the invention the following substitutions are
considered as conservative: replacement of arginine by cytroline; arginine by
glutamine; aspartate by asparagine; glutamate by glutamine.

25 As the naturally occurring amino acids are grouped according to their

properties, conservative substitutions by naturally occurring amino acids can be
easily determined bearing in mind the fact that in accordance with the invention
replacement of charged amino acids by sterically similar non-charged amino acids
are considered as conservative substitutions.

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For producing conservative substitutions by non-naturally occurring amino
acids it is also possible to use amino acid analogs (synthetic amino acids) well
known in the art. A peptidomimetic of the naturally occurring amino acid is well
documented in the literature known to the skilled practitioner.
5 The following are some non-limiting examples of groups of naturally

occurring amino acids or of amino acid analogs are listed bellow. Replacement of
one member in the group by another member of the group will be considered herein
as conservative substitutions:

Group I includes leucine, isoleucine, valine, methionine, phenylalanine,
10 serine, cysteine, threonine and modified amino acids having the following side
chains: ethyl, n-butyl, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 OH, -CH 2 CHOHCH 3 and-
CH 2 SCH 3 . Preferably Group I includes leucine, isoleucine, valine and methionine.

Group II includes glycine, alanine, valine, serine, cysteine, threonine and a
modified amino acid having an ethyl side chain. Preferably Group II includes
15 glycine and alanine.

Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan,
cyclohexylmethyl, and modified amino residues having substituted benzyl or
phenyl side chains. Preferred substituents include one or more of the following:
halogen, methyl, ethyl, nitro, methoxy, ethoxy and -CN. Preferably, Group III
20 includes phenylalanine, tyrosine and tryptophan.

t

Group IV includes glutamic acid, aspartic acid, a substituted or unsubstituted
aliphatic, aromatic or benzylic ester of glutamic or aspartic acid (e.g., methyl, ethyl,
n-propyl iso-propyl, cyclohexyl, benzyl or substituted benzyl), glutamine,
asparagine, CO-NH-alkylated glutamine or asparagine (e.g., methyl, ethyl, n-propyl
25 and iso-propyl) and modified amino acids having the side chain -(CH 2 ) 3 COOH, an
ester thereof (substituted or unsubstituted aliphatic, aromatic or benzylic ester), an
amide thereof and a substituted or unsubstituted N-alkylated amide thereof.
Preferably, Group IV includes glutamic acid, aspartic acid, glutamine, asparagine,

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methyl aspartate, ethyl aspartate, benzyl aspartate and methyl glutamate, ethyl
glutamate and benzyl glutamate.

Group V includes histidine, lysine, arginine, N-nitroarginine,
P-cycloarginine, }i-hydroxyarginine, N-amidinocitruline and

5 2-amino-4-guanidinobutanoic acid, homologs of lysine, homologs of arginine and
ornithine. Preferably, Group V includes histidine, lysine, arginine, and ornithine. A
homolog of an amino acid includes from 1 to about 3 additional methylene units in
the side chain.

Group VI includes serine, threonine, cysteine and modified amino acids
10 having C1-C5 straight or branched alkyl side chains substituted with -OH or -SH.
Preferably, Group VI includes serine, cysteine or threonine.

The term "non-conservative substitutions" concerns replacement of one or
more amino acid residues present in the original molecule by another naturally or
non-naturally occurring amino acid, having different a different size, configuration
1 5 and/or electronic properties compared with the amino acid being substituted. Thus,
the side chain of the substituting amino acid can be significantly larger (or smaller)
than the side chain of the original amino acid being substituted and/or can have
functional groups with significantly different electronic properties than the amino
acid being substituted.

20 According to the invention modification may also involve non-conservative

substitutions, as long as that the immunological activity of the amino acid molecule
is retained.

"Peptidomimetic organic moiety" can be substituted for amino acid residues
in the molecules of the invention both as conservative and as non-conservative
25 substitutions. The peptidomimetic organic moieties often have steric, electronic or
configurational properties similar to the replaced amino acid.

Peptidomimetics are often used to inhibit degradation of the amino acid
molecules by enzymatic or other degradative processes. The peptidomimetics can
be produced by organic synthetic techniques. Examples of suitable peptidomimetics
30 include D amino acids of the corresponding L amino acids, tetrazol [Zabrocki et al,

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J. Am. Chem. Soc. 110:5875-5880 (1988)]; isosteres of amide bonds [Jones et al,
Tetrahedron Lett. 29: 3853-3856 (1988)]; LL-3-amino-2-propenidone-6-carboxylic
acid (LL-Acp) [Kemp et al, J. Org. Chem. 50:5834-5838 (1985)]. Similar analogs
are shown in Kemp et al, Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp
5 et al, Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al, Tetrahedron Lett.
29:4935-4938 (1988) and Kemp et al, J. Org. Chem. 54:109-115 (1987). Other
suitable peptidomimetics are shown in Nagai and Sato, Tetrahedron Lett. 26:647-
650 (1985); Di Maio et al, J. Chem. Soc. Perkin Trans., 1687 (1985); Kahn etal,
Tetrahedron Lett. 30:2317 (1989); Olson et al, J. Am. Chem. Soc. 112:323-333
10 (1990); Garvey et al, J. Org. Chem. 56:436 (1990). Further suitable
peptidomimetics include hydroxy- 1,2,3,4-tetrahydroisoquinoline- 3-carboxylate
(Miyake et al, J. Takeda Res. Labs 43:53-76 (1989)); 1,2,3,4-tetrahydro-
isoquinoline-3-carboxylate [Kazmierski et al, J. Am. Chem. Soc. 133:2275-2283
(1991)]; histidine isoquinolone carboxylic acid (HIC) [Zechel et al, Int. J. Pep.
15 Protein Res. 43 (1991)]; (2S, 3 S)-methyl-phenylalanine, (2S, 3R)-methyl-
phenylalanine, (2R, 3S)-methyl- phenylalanine and (2R, 3R)-methyl-phenylalanine
[Kazmierski and Hruby, Tetrahedron Lett. (1991)].

"Deletion" includes exclusion of one or more amino acid residues
(naturally occurring, non-naturally occurring, or peptidomimetic organic moiety)
20 as compared to the original molecule from which it is derived.

"Insertion " or "addition " includes the addition of one or more amino
acid residues (naturally occurring, non-naturally occurring, or peptidomimetic
organic moiety) as compared to the original molecule from which it is derived.

"Chemical modification" includes modification at the side chain of the
25 amino acid residue, as well as modification of the peptidic bond. Accordingly, a
functional group may be added to the side chain, deleted from the side chain or
exchanged with another functional group. Typically, the modifications are
conservative modifications resulting in conservative substitution. Examples of
conservative modifications of this type include adding an amine or hydroxyl,
30 carboxylic acid to the aliphatic side chain of valine, leucine or isoleucine,

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exchanging the carboxylic acid in the side chain of aspartic acid or glutamic acid
with an amine or deleting the amine group in the side chain of lysine or ornithine.

Other chemical modifications known in the art include arboxymethylation,
acylation, phosphorylation, glycosylation or fatty acylation, and others.

5 According to the invention, the modification should be an immunological

modification, i.e. such that the antigenic binding characteristic or the
immunostimulatory effect of the modified amino acid molecule (i.e. the
immunogenecity) is substantially retained as compared to the original amino acid
molecule from which they are derived.

10 "Carrier proteins" are known in the art. It is acceptable that small

molecules such as drugs, organic compounds, and peptides and oligosaccharides
with a molecular weight of less than 2-5 kDa are less immunogenic, even when
administered in the presence of adjuvants. In order to generate an immune
response to these compounds, they are typically attached to a carrier protein or

15 other carrier compound that is immunogenic. The conjugation of the carrier
protein to the amino acid molecule of the invention may result in stimulation of
an immune response to the peptide (cellular as well as humoral response) as well
as in an increase in the intensity of the immune response to the peptide [Jacobson
RM, et al. Minei*va Pediatr. 54(4):295-303 (2002)].

20 Non-limiting examples of carrier proteins which may be utilized

according to the invention include Keyhole limpet hemocyanin (KLH); Bovine
serum albumin (BSA); Rabbit serum albumin (RSA); Ovalbumin (OVA);
Pumpkin Seed Globulin (PSG), tetanus toxoid (TT), diphtheria toxin (CRM).

Other carrier proteins which may be utilized in accordance with the
25 invention include dentritic polymers (dendrimers, dendrons and hyperbranched
dendritic polymers) which are synthetic, three-dimensional macromolecule. The
dendritic polymers are built up from monomers, with new branches added in
steps until a tree-like structure is created: When using dendritic polymers as a

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carrier, multiple copies of the same or different amino acid molecule according to
the invention may be conjugated to each branch of the tree-like structure.

According to one preferred embodiment, the amino acid molecule
comprises Bll peptide (fSLTEWSMWYRH, SEQ ID NO:l), and conservative
5 modifications thereof. One example of a conservative modification includes the
replacement of Try (W) with another aromatic amino acid residue, such as Phe
(F), His (H) or Tyr (Y).

The peptides identified above and which form part of the amino acid
molecule according to the invention bind with specificity to ManLAM binding
10 antibodies, preferably to anti-ManLAM antibodies.

As described hereinabove, ManLAM is a major cell surface component of
virulent Mycobacterium sp. In several publications it has been shown that sera of
Tuberculosis patients contain higher levels of ManLAM binding antibodies than
that of healthy individuals [Hetland G, et al. (1998) ibid.].
15 Thus, according to the invention there is provided a method for

diagnosing a mycobacterial infection in a subject by determining the presence of
ManLAM binding antibodies in a sample from said subject. According to one
embodiment, the ManLAM binding antibodies are anti-ManLAM antibodies.
The diagnostic method of the invention comprises contacting the sample with the
20 amino acid molecule and determining formation of a complex between the amino
acid molecule and ManLAM binding antibodies. The formation of such a
complex indicating that said subject is or has been infected by a mycobacterial
agent.

In the context of the present invention the term "subject" denotes mammal
.25 subjects, including humans and non-human animals, such as cattle, sheep, deers,
dogs, cats etc.

"Sample" denotes a fluid bodily sample, preferably a blood sample
(including whole blood or blood's serum) or a bodily secretion, e.g. urine, saliva,
phlegm etc.

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Tfre method of the present invention may be by binary (yes/no)
determination of the presence of ManLAM binding antibodies in the serum.

However it was also surprisingly found that the amino acid molecules of
the invention are capable of distinguishing between individuals having acute
5 infection of TB and individuals immunized for TB. This finding leads the way to
a method of determining active infection of TB (as compared to existence of anti-
TB antibodies present in the individual as a result from prior immunization) by
the determination of the levels of the complexes between the amino acid of the
invention and antibodies.
10 Thus the present invention concerns a method for determining whether a

subject has active mycobacterial infection, the method comprising:

(a) contacting a sample from said subject with an amino acid molecule
comprising a peptide comprising at least one of the following
characteristics:

15 i) being capable of binding to ManLAM-binding antibodies;

ii) being capable of eliciting, upon immunization in a subject,
production of ManLAM binding antibodies .

(b) determining level of complexes comprising said amino acid molecule
and ManLAM binding antibodies;

20 (c) comparing said level to a standard,

a level higher than said standard indicating active myobacterial infection in

the subject.

The determination may be by comparing the level of complexes formed in
a sample from a tested individual, to a pre-defined standard (threshold) obtained
25 by determining the level of complexes in clinically diagnosed active TB patients
as compared to individual immunized with TB but not actively ill.

The standard is a level wherein above it the person has a statistically
significant probability of falling under the TB-ill patients group and bellow it has
a statistically significant probability of falling under TB-immunized individuals

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(although there may be overlap between the actual levels). The standard may be
determined by statistical methods known in the art.

"Standard" denotes either a single standard value or a plurality of
standards with which the level of ManLAM binding antibodies from the tested

5 sample is compared. The standards may be provided, for example, in the form of
discrete numeric values or is colorimetric in the form of a chart with different
colors or shadings for different levels of antibodies; or they may be provided in
the form of a comparative curve prepared on the basis of such standards. The
standards may be prepared by determining the level of antibodies present in

10 samples obtained from a plurality of subjects. For example, the standards may be
determined by the level of antibodies in positive and negative Bacillus Calmette-
Guerin (BCG) vaccinated subjects and non-vaccinated healthy subjects.

The determination step may be performed using any of the technologies
available in the art. Non-limiting examples include visualization of the bound

15 amino-acid molecule-antibodies by the use of the relevant label conjugated
secondary antibody (e.g. molecular probes) which may be fluorescent, enzyme-
linked to give a colorimetric reaction (by enzyme-linked immunosorbent assay
(ELISA)), radioimmunoassay (RIA), by the use of a fluorescently labeled affinity
purification of the antibodies, (relevant to testing of cultures), FACS analysis,

20 Dot-Blot, confocal laser scanning microscopy (CLSM).

The determination step may also include the visualization of the bound
amino-acid molecule-antibodies by agglutination methods, e.g. by the use of
latex beads having immobilized onto their surface the amino acid molecules of
the invention; or by any other means, as known to those versed in the art.

25 In addition, it is possible by the method of the invention to determine

treatment efficacy by following the level of ManLAM binding antibodies in a
sample from a subject undergoing treatment for a mycobacterial infection. The
method comprises the steps of:

(a) obtaining samples from said subject, from at least two discrete time

30 points, and contacting the samples with an amino acid molecule

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comprising a peptide comprising at least one of the following
characteristics:

i) being capable of binding to ManLAM-binding antibodies;

ii) being capable of eliciting, upon immunization in a subject,
5 production of ManLAM binding antibodies.

(b) determining level of complexes comprising said amino acid molecule
and ManLAM binding antibodies in said samples;
wherein a difference in the level between the two time points is indicative
of the effectiveness of the treatment.
10 The time points may be before, during and after the treatment. For

example, a first sample may be taken at a time point prior to initiation of the
treatment and a second sample may be taken at a time point after the treatment. A
decrease in the level of complexes in the second sample as compared to the first
sample would be indicative that the treatment is effective.
15 In another example, a first sample may be taken at a time point during the

treatment and a second sample may be taken at a time point after the treatment. A
decrease in the level of the complexes in the second sample as compared to the first
sample would also be indicative that the treatment is effective.

This method is based on earlier evidence showing that levels of anti-
20 Glycolipid antibodies change post treatment [Maekura, R., et al., J Clin
Microbiol. 39(10):3603-8 (2001)].

According to one embodiment, the mycobacterial infection is an infection
caused by Mycobacterium tuberculosis (Mtb), Mycobacterium africanum or
Mycobacterium bovis (the tuberculosis complex), or by Mycobacterium leprae.
25 According to a preferred embodiment the mycobacterial infection is an

infection caused by Mycobacterium tuberculosis, thereby causing Tuberculosis.

The invention also provides a diagnostic assay kit for diagnosing
mycobacterial infection in a subject, the kit comprising one or more amino acid
molecule according to the invention. As typical for medical or diagnostic use, the
30 kit may also comprise instructions for use of the amino acid molecule for

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diagnosing the infection. The kit may also comprise other diagnostic
components, such as the substrate onto which the amino acid molecule is
immobilized (e.g. for ELISA), secondary antibodies (labeled) etc.

The specific experimental results presented herein provide evidence that

5 the amino acid molecules of the invention are capable of eliciting production of
antibodies which bind to ManLAM (specific, direct as well as competitive
binding). Since ManLAM is a unique fingerprint for Mycobacterium sp. it may
also be used as a vaccine for immunizing subjects against such infectious agents.
Thus, the invention also provides a vaccine comprising an

10 immunologically acceptable carrier and as an active agent amount of an amino
acid molecule according to the invention. The amount of the amino acid
molecule is determined such that an immunological response is produced upon
administration of the vaccine to the subject.

"Immunological response" in the context used herein denotes the
15 production of a protective immune response. Particularly, that the immune
response evoked by the amino acid molecule in question protects the subject
immunized from contracting a mycobacterial infection, or that the immune
response evoked by the amino acid molecule at least confers a substantially
increased resistance to infections with mycobacteria. The immune response
20 preferably includes the generation of antibodies that bind to mannosylated
glycans, with preference to ManLAM.

The vaccine may also include immunologically acceptable adjuvants,
Accordingly, immunization may be carried out in combination with whole phage
particles [Benhar L, Biotechnol Adv 19:1-33 (2001) ; Alum; Monophosphoryl
25 lipid A (MPL), CpG Islands, Saponins, cytokines, as well as commonly known
antigen delivery vehicles, such as emulsions, micro-particles, iscoms, liposomes,
virosomes and virus like particles [Singh M and Srivastava I. Curr HIV Res.
l(3):309-20 (2003)].

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The amount of the amino acid molecule in the vaccine is determined by such
considerations as may be known in the art. The amount must be effective to achieve
the desired immunological response as described above, i.e. production of
antibodies which bind to ManLAM to a level sufficient to achieve protection

5 against a threatening infection. This effective amount is typically determined in
appropriately designed trials (e.g. dose range studies) and the person versed in the
art will know how to properly conduct such trials in order to determine the effective
amount. Some considerations for determining the effective amount include the
clinical condition of the subject, the site and method of administration, scheduling

10 of administration, patient age, sex, body weight and other factors known to medical
practitioners.

The vaccines are administered in a manner compatible with the dosage
formulation, and in such amount as will be immunogenic. The quantity to be
administered depends on the subject to be treated, including, e.g., the capacity of
1 5 the individual's immune system to mount an immune response, and the degree of
protection desired. Suitable regimes for initial administration and booster shots
are also variable but are typified by an initial administration followed by
subsequent inoculations or other administrations.

The vaccine may comprise one or more amino acid molecules of the
20 invention, or alternatively, the amino acid molecule may comprise multiple
copies of the same or different peptides as defined hereinabove. A preferred
vaccine is such that the amino acid molecule comprises a carrier protein linked to
one or more peptides as defined herein.

According to one embodiment, the vaccine is administered to the subject
25 in need by means of parenteral administration, e.g. intravenously (i.v.),
intradermally (i.d.) intraperitoneally (i.p.), intramuscularly (i.m.), subcutaneous
(s.c), intranasal (i.n.), intrarectally as well as by oral route or by inhalation.

The invention also provides a method of immunization of a subject against
mycobacterial infection; the method comprises providing said subject with an
30 amount of an amino acid molecule as defined, the amount being sufficient to

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achieve effective immunity effect against infectious agents carrying mannose-
containing cell-wall lipoglycans, such as ManLAM, LM and AM.

As will be appreciated by those versed in the art, the subject may be
provided with a single dose or multiple doses of the immunogenic amino acid
5 molecule of the invention. The multiple doses may be provided over an extended
period of time in a single daily dose or in several doses a day. The doses and
schedule of treatment is determined by considerations available to those known
in the art.

SPECIFIC EXAMPLES
10 Materials and Methods

Biopanning of phage display libraries

Phage display libraries were constructed in the fthl type 88 vector
[Enshell-Seijffers D, and Gershoni, J.M. epitopes, In Current Protocols in
Immunology, Vol. 2. J.E. Coligan, ed. Wiley, New York. p. 9.8.1(2002)]; Stern,
15 B., et al. Faseb J 11, 147-153 (1997)] at the laboratory of Prof. Gershoni
(Department of Cell Research and Immunology, George S. Wise Faculty of Life
Sciences, Tel Aviv University, Israel)

Biopanning of random phage display libraries was performed as described
in Enshell-Seijffers and Gershoni [Enshell-Seijffers and Gershoni (2002), ibid.],
20 based on the methods of Smith and Scott, [Smith, G. P., and Scott, J. K. Methods
Enzymol 217:228-257 (1993)]. Briefly, 6-well polystyrene plates (Nunc,
Roskilde, Denmark) were coated with mAbs (5 ug/well) in TBS and then
blocked with TBS/0.25% gelatin for 2 h at RT. Phage particles (lxlO 11 ) in
TBS/0.25% gelatin were bound to the mAb-coated wells at 4°G for 16 h. The
25 unbound phages were removed by 4 washes of TBS, and the bound phages were
eluted by adding a glycine/HCl/BSA buffer, pH 2.2, for 10 min, which was then
neutralized using Tris buffer, pH 9.1. Phages were amplified in Escherichia coli
DH5ct. For titer determination, aliquots of the eluate or the amplificate were

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plated in serial dilutions on Luria broth (LB) agar plates. Up to six biopanning
rounds were performed

Phage clone screening assay and peptide sequencing

Screening of the phage clones tested for recognition with the CS40 anti-

5 ManLAM mAb was performed by the immuno Dot-Blot technique (Enshell-
Seijffers D. et al., J Mol Biol, 334, (2003); Enshell-Seijffers and Gershoni,
(2002) ibid.]. Briefly, the phage particles were bound directly to nitrocellulose
membranes (Schleicher and Schuell GmbH, Dassel, Germany), which were then
blocked with TBS/10% skim milk (1 h at RT). The anti-polysaccharide mAb was

10 added (1 ug/ml) to me membranes and incubated overnight at 4°C. The
membranes were then washed in TBS 5 times for 5 min at RT. The secondary
antibody (HRP-conjugated goat anti-mouse IgG (Jackson Laboratories, West
Grove, PA) diluted 1:5000 was then added (incubated for 1 h at RT). Positive
clones were detected by Dot-Blot. ManLAM was used as a positive binding

15 control. Phage clones that were determined to be positive by the Dot-Blot assay
were retested by the same method in triplicate and by ELISA competition. In the
selection of the phages with each of the Abs, a control phage clone was selected.
These control phage clones did not bind the relevant mAb and did not present
amino acids of the motif. The control phages were used as negative controls in

20 binding and immunization assays.

Single-stranded DNA of the positive phage clones was isolated using the
QIAprep Spin Ml 3 Kit® (QIAGEN, Hilden, Germany). The DNA sequences
encoding the peptide insert were analyzed at the Hebrew University, Jerusalem,
Israel by an automated ABI 310 DNA sequencer (Perkin-Elmer, Santa Clara,
25 CA). The deduced peptide sequences were aligned by ClustalW alignment using
MacVector™ 7.0 (Oxford, UK). The anti-glucuronoxylomannan (GXM) mAb
2H1 was obtained from Prof. Casadaval (Albert Einstein College of Medicine,
New York). The anti-MgS sugar mAb 735 was obtained from Prof. Bitter-
Suermann (Institute of Medical Microbiology, Hannover, Germany). ManLAM

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from M. tuberculosis strain H37Rv, and anti-ManLAM mAb (CS40), were
obtained from Drs. Brennan and Belisle (Colorado State University, Fort Collins,
CO).

Direct peptide and ManLAM ELISA assays
5 Direct ELISA assays were performed by coating 96-well ELISA plates

(O/N at 4°C) with 5-20 u.g peptide per well in PBS ManLAM 5 ug/well
(50 ul/well). The plates were washed twice in phosphate buffered saline (PBS),
blocked with PBS/1% BSA and then washed twice (PBS). Sera diluted in
PBS/0.5% BSA (1:50 or 1:200) or CS40 mAb (1:2000) (50 ul per well) was
10 added and incubated for 1 h at 37°C. After washing, alkaline phosphatase-labeled
goat anti-mouse immunoglobulin (Sigma, St. Louis, MO) was added. Following
90-min incubation at 37°C, p-nitrophenyl phosphate (KPL, Gaitherburg, MD)
was added to the plates, and optical density was measured at 405 nm using
ELISA reader (ELX-800UV, Bio-Tec instruments, Winooski, VT). In all ELISA
1 5 procedures, samples were tested in triplicate.

Human sera samples, after appropriate Helsinki endorsement, were
obtained from Dr. Rahav (Chaim Sheba Hospital, Ramat Gan, Israel), Dr.
Spectre (Hadassah University Hospital, Jerusalem, Israel), and Prof. Marchal
(Pasteur Institute, Paris).

20 ELISA competition

ELISA competition assay was performed according to Kaur et aL, [Kaur,
D., et al. Microbiology 148:3049-3057 (2002)]. Briefly, plates were coated with
0.5 jig/well ManLAM. Plates were blocked as described above. The CS40 mAb
(diluted 1 :2000) was mixed with various concentrations of either phage particles
25 in TBS/1% BSA (0-lxlO 18 particles/well) or peptides in PBS/1% BSA (0-200
ug/well). This solution was then transferred to the ELISA plates and incubated
for 1 h at 37 °C. After washing (PBS), the ELISA procedure was performed as
described above. Samples were all tested in triplicate

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Phage immunization

Phage immunization was performed by subcutaneous (s.c.) injection to
mice of the selected phage clones, lxl 0 9 - lxl 0 13 phage particles/mouse, or a
control phage (as described above) in MPL-TDM adjuvant system (Sigma). The
5 mice were boosted with the same dose three weeks later. Three weeks after the
last immunization, production of specific polysaccharide binding IgG Abs was
assessed in sera obtained by bleeding the tail vein of the immunized mice.

Peptide synthesis and conjugation

Peptides were synthesized by a solid-phase technique. For binding assays,
10 peptides corresponding to the phage clones (Al, Bl 1) were synthesized with no
additional amino acids. For immunization, the Bll peptide was synthesized with
an additional cysteine residue at the amino terminus for conjugation to
sulfhydryl-reactive KLH, performed according to manufacturer's instructions
(Pierce, Rockford, IL)
15 Peptide synthesis was performed by Prof. Fridkin at the Weizmann

Institute, Rehovot, Israel, and at the Interdepartmental Facility of the Hebrew
University Faculty of Medicine, Jerusalem, Israel.

Peptide immunization

Bll (5 ug and 50 ug) conjugated peptides were administrated to specific
20 pathogen-free (SPF) female BALB/c mice, 5-8 weeks old, intraperitoneally (i.p.)
or s.c. The mice were boosted with the same dose three weeks later. In the s.c.
immunization route, various adjuvants were tested: KLH-conjugated peptide was
emulsified in MPL-TDM adjuvant system (Sigma) or in incomplete Freund's
adjuvant (IF A) (Sigma) according to manufacturer's instructions, or in
25 dimethyldioctadecylammonium bromide (DDA) (Fluka, Buchs, Switzerland).
Immunization i.p. was performed using the KLH-conjugated peptide emulsified
in the MPL- TDM adjuvant system. In all immunization experiments, control
groups were immunized with KLH in the relevant adjuvant with no peptide.

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In all immunized groups production of Abs was tested in the sera. For this,
mice were bled from the tail vein at three times: pre-immunization, two weeks
after first administration, and three weeks after second administration. All
experiments were performed in accordance with the regulations of the animal
5 experimentation ethics committee of the Hebrew University-Hadassah Medical
School.

4

Experimental Mycobacterium tuberculosis infection

SPF female BALB/c mice were inoculated i.v. in the tail vein with 5x10
CFU of M. tuberculosis strain H37Rv in 200 ul of saline (a kind gift from Prof.

10 G. Marchal, Pasteur Institute, Paris). Thirty days (6 mice) and three months (4
mice) after Mtb infection the mice were bled and tested for the presence of IgG
antibodies that bound ManLAM and Bll peptide. The sera of six uninfected
mice were used as negative controls. All experiments were performed in
accordance with the regulations of the animal experimentation ethics committee

1 5 of -the Hebrew University-Hadassah Medical School.

Cell culture

The cell, line RAW 264.7 obtained from the American Type Culture
Collection was grown in Dulbecco's modified Eagle's medium (DMEM)
. supplemented with 10% FCS, 1% penicillin, 1% L-glutamine, 1% nonessential
20 amino acids, and 1% pyruvate (Biological Industries, Beit Haemek, Israel) to an
80% confluent culture at 37°C in an atmosphere enriched with 5% C0 2 .

Confocal microscopy of FITC-labeled Bll peptide binding to RAW 264.7
cell line

Bll peptide (50 ug) was conjugated to FITC (Biotium, Hayward, CA) as
25 described by Sethi et al., [Sethi, K. K., et al. Cell Motil Cytoskeleton 52:23 1-241
(2002)]. For confocal microscopy, RAW 264.7 cells were grown in 8-well glass
slides, Chamber Slide™ (Nunc), as described above. Cells were washed three
times in cold PBS and incubated with the FITC-labeled mimotope for 5 min at

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4°C in serial dilutions of 1:2-1:100 in cold PBS^ Slides were imaged immediately
with a Zeiss 410 confocal laser scanning microscope.

FACS analysis of FITC-labeled mimotope binding to RAW 264.7 cell line

RAW 264.7 cells were grown in a 250-ml flask, washed 3 times in cold
5 PBS, and harvested. Cells were then incubated for 15 min at 37°C, with the
FITC-labeled peptide diluted 1:50, then washed 3 times in cold PBS. The
fluorescence levels of 10,000 cell counts were determined by FACS (Becton
Dickinson, Franklin Lakes, NJ) and analyzed by CELLQuest™ software. Assay
of competitive binding between mannose and the FITC-labeled B 1 1 peptide was
10 performed according to Nguyen and Hildreth, [Nguyen, D. G., and Hildreth, J. E.
Eur J Immunol 33:483-493 (2003)].

RESULTS

Sequences selection by phage display technology

In the search for mimotopes of mannosylated lipoarabinomannan
15 (ManLAM), a major cell surface component of virulent Mycobacterium, phage
display libraries were screened with CS40, a mAbs obtained against ManLAM
from Mycobacterium Tuberculosis (Mb) [Chatterjee, D., et al.,. J Biol Chem
267:6234-6239, (1992)], as described in Material and Methods. CS40 was shown
to bind mannosylated molecules ManLAM and arabinomannan (AM). The
20 specific epitope bound by is not yet defined [Navoa, J. A.,et al. Clin Diagn Lab
Immunol 10:88-94 (2003)].

All selected phage clones were tested by Dot-Blot for specific binding to
the anti-ManLAM CS40 mAb. All phage clones bound only to the CS40 mAb
and not to three other anti-polysaccharide mAbs: CS35 anti-LAM mAb [Kaur et
25 al. (2002). ibid.], 735 anti-ploy a(2-»8) N-acetyl neuraminic acid mAb
[Kibbelaar, R. E., et al. J Pathol 159:23-28 (1989)], and 2H1 anti-
glucuronoxylomannan mAb [Mukherjee, J., et al. Infect Immun 60:4534-4541
(1992)].

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As is illustrated in Table I, 6 different phage clones were isolated with
mAb CS40. In five of the clones a core motif of 4-5 residues in which a central
tryptophan is flanked by hydrophilic amino acids (E/RWS/EXH/K) was
observed.

10

Table 1: ManLAM peptide mimotopes

done Amino acid sequence

Al

WEADDKNQHGEG

(SEQ ID NO:6)

Bll

ISLTEWSMWYRH

(SEQ ID NO: 1)

CI

EEGPWSTHVGRT

(SEQ ID NO:2)

C3

WGNEGGDHLQPV

(SEQ ID NO:3)

F7

SLKIKWELKMYQE

(SEQ ID NO:4)

G3

AVEKWEKHTWSE

(SEQ ID NO: 5)

*The postulated motif amino acids are indicated in gray box and bold letters

1 5 Binding properties of the phage clones and synthetic peptides (Bll and Al)

Efficiency of binding was evaluated by the magnitude of OD decrease
when serial dilutions of phage clones were incubated with the CS40 mAb. The
phage clones with the higher affinity to mAb showed maximum inhibition of the
binding of the mAb to ManLAM with the lowest number of phage particles.
20 When testing the clones that bound the CS40 mAb, two clones, Bll and CI,
competed most efficiently with ManLAM (Fig. 1A); 1 X 10 11 phage particles per
well reduced the OD by 100%. While with clone G3, a similar reduction in Hie
OD was measured, however, it was obtained with 1X1 0 14 phage particles per
well.

25 The synthetic Bll peptide bound the CS40 mAb, as tested by direct

ELISA, and competed with ManLAM in binding to the mAb, as tested in
competitive ELISA (100% decrease in OD with 50 ug/ml of peptide (Fig. IB).
The competitive binding properties were compared to those of a peptide
corresponding to the sequence of clone Al. Al peptide did not compete in the

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binding of the antibodies vs. ManLAM, thus indicating that the internal aromatic
residue and adjacent hydrophilic residues present in the other mimotopes, such as
in Bl 1 peptide, are important for the binding to the CS40 mAb.

Antigenic properties of Bll phage clone and synthetic peptide

5 Bll phage clone was tested for the ability to induce ManLAM-binding

Abs in sera of immunized mice. Mice (n=5) injected twice with the Bll phage
clone developed IgG antibodies specific to ManLAM, compared to naive mice
and mice immunized with a control phage, as demonstrated by a serological
ELISA test (Fig. 2A).

10 The antigenic properties of the Bll synthetic peptide was tested for the

ability to induce IgM and IgG ManLAM binding Abs. This was performed by
immunizing groups of five mice (n=5 per group) with the B 1 1 synthetic peptide
conjugated to KLH via an extra cystiene residue at the N-terminus. Significant
levels of IgM Abs, which bound specifically to ManLAM, were detected in mice

15 vaccinated s.c. with 50 ug peptide conjugated to KLH emulsified in the MPL-
TDM adjuvant system, two weeks after the first immunization (p<0.05 vs. the
control groups) (Fig. 2B). In the group vaccinated s.c. with 50 ug peptide
conjugated to KLH in MPL'TDM, no significant levels of IgG were found after
the first immunization. Significant levels (p<0.05 vs. the control groups) of

20 specific ManLAM-binding IgG Abs were detected three weeks after the second
s.c. immunization (Fig. 2C). The IgGs were mostly of the subtype IgGl (Fig.
2D). These results were reproduced in three separate experiments. No significant
ManLAM-binding antibody levels were detected in any of the other immunized
groups (data not shown).

25 Covalently conjugating the polysaccharide antigen to a carrier protein was

used as it improves the immune-response by permitting the host to utilize a T-cell
dependent immune response [Jacobson, R. M., and Poland, G. A. Minerva
Pediatr 54 :295-3 03 (2002)] .

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IgG antibodies that bind to the Bll synthetic peptide in M^-infected mice

To further investigate if the Bll peptide is a true mimotope of the
ManLAM, mice experimentally infected with Mtb that have never been exposed
to the peptide were tested for development of antibodies that recognize the B 1 1

5 peptide, similar to the Abs developed against ManLAM. To this end, thirty days
(n=6) and three months (n=4) after an experimental Mtb infection, sera of
BALB/c mice were tested for the presence of IgG that recognized ManLAM and
Bll peptide, and were compared to naive mice (n=6). In the Mtb-infected mice
IgG Abs which binds both ManLAM and B 1 1 peptide were detected, at levels

10 significantly higher than those of the naive mice (pO.Ol). The antibody levels
binding ManLAM as well to Bll peptide were similar (Fig. 3). The same results
were obtained when the ELISA assay was performed with or without the extra
cysteine at the amino teriminus of the synthetic peptide (data not shown). This
gave additional evidence that the peptide with the cysteine

15 (CI SLTE WSMWYRH) maintained binding properties to ManLAM-binding
antibodies as the original peptide selected (ISLTEWSMWYRH).

IgG peptide-binding antibodies in human tuberculosis patients

High levels of anti-ManLAM antibodies were measured in tuberculosis
(TB) patients [Hamasur, B., et al. J Microbiol Methods 45:41-52 (2001)]. The

20 presence of serum antibodies in sera of TB patients that bind the B 1 1 peptide was
tested. Significantly higher titers of. ManLAM and Bll peptide-binding
antibodies in sera of TB patients (n=16) than in sera of healthy individuals
(n=36) were found (Fig.4). Like the antibody levels measured in mice, the human
anti-ManLAM and Bll peptide-binding antibody levels were similar, but the

25 variability of the serum titers (SD=0.129 for ManLAM vs. SD=0.058 for the
peptide) was smaller when using the Bl 1 peptide as an antigen in the ELISA test
(Fig. 4). This might indicate that the peptide can be a more reliable reagent than
ManLAM for serological diagnosis. Using ELISA, sera from Bacillus Calmette
Guerin (BCG)-vaccinated individuals (n=10) was tested, as it is sometimes

30 difficult to distinguish BCG-vaccinated individuals from TB patients [Ciesielski,

WO 2005/080414 PCT/IL2005/000199

- 32 -

S. D., J Fam Pract 40:76-80 (1995)]. The anti-peptide antibody titers measured
in BCG-vaccinated individuals were significantly lower than in TB patients
(pO.OOl) (Fig. 4). Standard deviation (SD) values were smaller in all groups
when using the Bl 1 peptide as an antigen compared to the SD values in binding

5 ManLAM as an antigen. SD values of Bll peptide coated plates: Non-
vaccinated-SD=0.035, Vaccinated- SD= 0.058 TB patients-SD= 0.05874. SD
values of ManLAM-coated plates: Non-vaccinated-SD=0.057, Vaccinated- SD=
0.084, TB-patients-SD=0. 129.

Thus, use of the B 1 1 peptide and the like, as defined herein, as the antigen

10 in an ELISA test provides a new and accurate tool to discriminate between TB
patients, healthy BCG-vaccinated and non-vaccinated individuals.

*

Bll peptide binds the murine macrophage cell line RAW 264.7

ManLAM binds macrophages via mannose-binding receptors [Maeda, N.,
et al. J Biol Chem 278:5513-5516 (2003)]; therefore, the binding of the peptide

15 mimotope of ManLAM to macrophages was examined. Indeed, the FITC-
conjugated Bll peptide bound the macrophage cell line RAW 264.7, as seen by
confocal microscopy (Fig. 5A) and FACS analysis (Fig. 5B). The binding of the
Bll peptide to the macrophages was inhibited by D-mannose, as detected by
FACS analysis (Fig. 5C), indicating that the peptide binds a macrophage

20 mannose receptor.